www.nature.com/scientificreports OPEN Population genetic analysis in old Montenegrin vineyards reveals ancient ways currently active to generate diversity in Vitis vinifera Vesna Maraš1, Javier Tello2, Anita Gazivoda1, Milena Mugoša1, Mirko Perišić1, Jovana Raičević1, Nataša Štajner3, Rafael Ocete4, Vladan Božović5, Tatjana Popović6, Enrique García‑Escudero2, Miodrag Grbić2,7,8, José Miguel Martínez‑Zapater2 & Javier Ibáñez2* Global viticulture has evolved following market trends, causing loss of cultivar diversity and traditional practices. In Montenegro, modern viticulture co‑exists with a traditional viticulture that still maintains ancient practices and exploits local cultivars. As a result, this region provides a unique opportunity to explore processes increasing genetic diversity. To evaluate the diversity of Montenegrin grapevines and the processes involved in their diversifcation, we collected and analyzed 419 samples in situ across the country (cultivated plants from old orchards and vines growing in the wild), and 57 local varieties preserved in a grapevine collection. We obtained 144 diferent genetic profles, more than 100 corresponding to cultivated grapevines, representing a surprising diversity for one of the smallest European countries. Part of this high diversity refects historical records indicating multiple and intense introduction events from diverse viticultural regions at diferent times. Another important gene pool includes many autochthonous varieties, some on the edge of extinction, linked in a complex parentage network where two varieties (Razaklija and Kratošija) played a leading role on the generation of indigenous varieties. Finally, analyses of genetic structure unveiled several putative proto-varieties, likely representing the frst steps involved in the generation of new cultivars or even secondary domestication events. Te cultivated grapevine (Vitis vinifera subsp. sativa L.) was likely frst domesticated from the wild grapevine (V. vinifera subsp. sylvestris (C.C.Gmel.) Hegi) in the Transcaucasian region (modern Georgia, Armenia, and Azerbaijan) about 8,000 years ago 1. Ten, early cultivars spread around the Mediterranean basin following the main human migration routes during several thousand years2. During this process, certain vines were selected for their adaptation to regional conditions and their ability to overcome local biotic and abiotic stresses. Tese vines become stable varieties through their vegetative propagation and evolved through combining mutation and sexual reproduction events with other early-domesticated cultivars or with wild autochthonous vines2. 113 Jul Plantaže, Radomira Ivanovića br. 2, 8100 Podgorica, Montenegro. 2Instituto de Ciencias de la Vid y del Vino (CSIC, UR, Gobierno de La Rioja), Ctra. de Burgos Km. 6, 26007 Logroño, Spain. 3Biotechnical Faculty, Agronomy Department, University of Ljubljana, Jamnikarjeva 101, 1000 Ljubljana, Slovenia. 4Laboratorio de Entomología Aplicada, Facultad de Biología, Universidad de Sevilla, Avenida de la Reina Mercedes s/n, 41012 Seville, Spain. 5Faculty for Food Technology, Food Safety and Ecology, University of Donja Gorica, 81000 Donja Gorica, Podgorica, Montenegro. 6Biotechnical Faculty, University of Montenegro, Mihaila Lalica 1, 81000 Podgorica, Montenegro. 7Department of Biology, University of Western Ontario, 1151 Richmond Street, London N6A5B7, Canada. 8Faculty of Biology, University of Belgrade, Studentski trg. 16, Beograd 11000, Serbia. *email: [email protected] SCIENTIFIC REPORTS | (2020) 10:15000 | https://doi.org/10.1038/s41598-020-71918-7 1 Vol.:(0123456789) www.nature.com/scientificreports/ From ancient times to modern days, the Western Balkans have been home of important European civili- zations. Illyrians, Greeks, Romans, Byzantines, Ottomans and Austro-Hungarians settled along this region, promoting the exchange of people, commodities and knowledge within close and distant regions (Near Asia, Dalmatia, Magna Graecia, etc.)3. First grapevines were introduced in the Balkan Peninsula from the East 4, with the earliest-known evidences of winemaking found in Northern Greece dating back to ca. 4,300 BCE 5. Ancient Greeks gradually spread grape varieties across the Western side of the Balkans and the Adriatic islands follow- ing main trading routes2,6, and Illyrians and other native populations progressively replaced the consumption of cereal-based beverages (mead, beer) with wine7. Winemaking and wine trading were two important activities for local economies at that time, as inferred from relics like those found in the necropolis of the coastal city of Budva (in modern Montenegro, dated from the fourth century BCE)8. Viticultural practices were intensifed by Romans afer the Illyrian Wars, as observed in Dionysian/Bacchic iconography, and in many remnants from domestic utensils used for wine transportation or consumption (pottery, amphorae, cups)7. Wine production continued during the Byzantine period, and documents like the Medieval Statute of Budva refects the impor- tance of grapevine cultivation in Montenegro in the Middle Ages. Te Statute, dated between 1,426 and 1,442, includes the relevance of certain grapevine varieties for local wine production 9,10, like the cultivar “Cratosia”, probably referring to the grape cultivar currently known as Kratošija (meaning short-neck in Montenegrin)9. Later, many vineyards were destroyed during the Ottoman period, and only some survived in remote regions in the center of the country and in several parts of the coastal regions controlled by Venice 6. Winemaking prac- tices were partially restored under the rule of Nikola I (1860–1918), which promoted wine exports to Western countries. Afer several millennia of genetic diversifcation, most of the grapevine genetic diversity generated in the Balkans was destroyed by the efect of phylloxera and mildews introduced from North America at the end of the nineteenth century2,11. As a result, many old autochthonous cultivars of the Western Balkans exist now as isolated plants or as relict populations, ofen represented by few specimens found in old, traditional vineyards or maintained in ex situ Vitis collections6,12, 13. Montenegro played a major role in the long history of grape cultivation in the Western Balkans. Its highly diverse climate conditions, isolated valleys, soil types and orography create highly diverse environments that, together with its historical and geographic context, promoted the generation of a rich grapevine biodiversity 14,15. Overall, grapes are grown in Montenegro over 2.800 ha, with a gross production of 22.200 t in 201716. Winemak- ing in Montenegro mainly relies on the production of red wine from two autochthonous grape cultivars, Vranac and Kratošija17. Kratošija was the dominant variety in the region until the phylloxera crisis, which forced the removal of many old vines and their replacement by new plant material 11. In many cases, withered Kratošija vines were replaced by grafed Vranac plants, preferred by growers as it produces highly deep-colored wines17. As a result, the Kratošija cultivated area decreased, while Vranac became the most commonly grown and the most emblematic cultivar in Montenegro during the twentieth century 14. Besides these two major cultivars, other less-known indigenous cultivars can be found in Montenegro and neighboring countries, including Bioka, Čubrica, Krstač and Žižak6,14,18. Worldwide wine consumption patterns have changed radically and rapidly in recent years, infuencing changes in viticulture. For a long time, grape growers selected and maintained local grape varieties to ft their viticultural practices and local climates, producing grapes that reached an appropriate concentration of sugars, acids and other compounds to make traditional wines 2. On the contrary, recent market globalization has caused a dramatic erosion in the diversity of wine grapes planted across the world, moving towards the cultivation of a reduced set of “international varieties”19. Tus, the exploration and analysis of winemaking regions where old vineyards are still maintained under traditional management practices can help to understand how European viticulture developed over time before the current standardization. As exemplifed in multiple regions13,18,20,21, wide surveys in traditional winemaking areas allow the identifcation of old genetic resources on the edge of extinction for their preservation and eventual exploitation. Grapes from unique native varieties can produce highly distinc- tive wines with real potential to revitalise local wine industries18,20,22, and there is a renewed global interest in local wine varieties from Old World nations to stand out in today highly standardized market. Furthermore, the analysis of these indigenous and traditional varieties has helped to successfully resolve previous doubts about cultivars parentage23–25, as well as to shed light on the likely origin and dissemination patterns of certain grapevine cultivars in traditional viticulture regions20,26. Te use of nuclear DNA markers, mainly microsatellites (or Simple Sequence Repeats, SSRs) and Single Nucleotide Polymorphisms (SNPs) have contributed decisively to the identifcation of grapevine varieties 27,28. Grape varieties are maintained through vegetative propagation, and thus all the plants of any variety present the same genotype for the molecular markers used29,30, while a few molecular markers are enough to distinguish any two varieties29,31. Tis fact has allowed to confrm and discover many